The growing interest in regenerative medicine has fueled the search for organ-specific stem or self-renewing cells. The best studied population of self-renewing cells is the hematopoietic stem cells (HSCs) as they are innovative options for treatment of diseases from cancer to metabolic disease to immunodeficiencies.
The process of blood cell formation whereby red and white blood cells are replaced through the division of HSCs located in the bone marrow is called hematopoiesis. The HSCs have the key properties of being able to self-renew and to differentiate into mature cells of both lymphoid and myeloid lineages. However, the genetic mechanisms responsible for the control of self-renewal and differentiation outcomes of HSC divisions remain largely unknown.
Currently, transplantation of human HSCs from adult bone marrow, mobilized peripheral blood, and umbilical cord blood (UCB) has been used clinically to treat hematopoietic cancers (leukemias and lymphomas) and to aid immune system recovery from high-dose chemotherapy of non-hematopoietic cancers. However, efficient transplantation requires substantial amount of HSCs from different sources and may require expansion.
HSCs can be originated from bone marrow, peripheral blood and UCB. The extraction of bone marrow cells requires surgery and painful procedure, and therefore becomes less favorable approach. Using peripheral blood cells is also a problem because the difficulty of obtaining qualified HSCs from the hematopoiesis compromised patient who suffer from illness or chemotherapy. UCBs are relatively easier obtained and the quality of HSCs is much higher, however, the number of HSCs obtained from this approach is still limited. Cell number from each extraction is sufficient for a child, but may be insufficient for an adult. To overcome this potential problem, a new approach that facilitates HSC proliferation in vitro by intervening stem cell self-renewal process is indeed necessary for HSC transplantation.
It has been indicated that transcription factors play a critical role in the regulation of gene expression and the differentiation in stem cells (Orkin, S. H. Nature Reviews Genetics 1, 57-64, 2000). Transcription factor switches various cellular processes through binding to specific gene target, and this regulation also depends on its cellular concentration. A group of transcription factors called DNA binding homeobox (HOX) was previously found to play a major role in embryogenesis. Recently, HOX family is also found to be involved in the development of HSCs (Buske, C. et. al., J. Hematol. 71, 301-308, 2000). The regulation of HSC self-renewal by HOX transcription factor was studied by Dr. Guy Sauvageau from the University of Montreal. Sauvageau's group showed that homeobox gene HOXB4 is critical in the regulation of HSC self-renewal for its ability of maintaining HSC population in bone marrow. HOX genes expressed in blood cells was first observed in human and mouse cell lines. Some types of HOX genes are expressed ubiquitously in various cell types, while others are specifically expressed in certain type of cells or certain time points during the development. For example, eight members of human HOXB cluster are expressed in early stage of erythrocyte development. However, HOXB genes such as HOXB4 and HOXB7 are also expressed in T cells and B cells. Sauvageau's group confirmed that nine HOXA, eight HOXB and four HOXC genes are expressed in CD34+ bone marrow cells. Among these CD34+ bone marrow cells, HOXB2, HOXB9 and HOXA10 are most enriched in erythrocyte progenitor cells. However, no HOX genes are expressed in CD34− cells. Human homeobox B4(HOXB4) gene was recently demonstrated to effectively expand HSCs in a retroviral or recombinant protein form. Recombinant TAT-HOXB4 proteins were used to expand stem cells in the laboratory scale without the risk of retroviral insertion or co-culture with bone marrow stromal cells (See Krosl, J. et al., Nature Medicine 9, 1428-1432, 2003). Therefore, HOXB4 protein is regularly used as a stimulant to promote HSCs expansion in vitro (FIG. 1).
Recent evidence indicated that by adding a TAT protein sequence tag at the N-terminus of HOXB4, exogenous HOXB4 can be delivered into the cell. This TAT sequence directs the transportation of HOXB4 from extracellular side into intracellular side. Upon entering the cytosol, HOXB4 can be refolded into its native conformation by chaperon HSP90. TAT-HOXB4 is able to promote HSC proliferation to 2-6 fold (Amsellem, S. et. al., Nature Medicine 9, 1423-1427, 2003; Krosl, J. et. al., Nature Medicine 9, 1428-1432, 2003). However, the yield of recombinant TAT-HOXB4 protein from E. coli by using regular purification procedure is too low.
In an effort to increase the yield of the recombinant TAT-HOXB4 protein, a method of making a TAT-HOXB4H protein with additional six histidine (SEQ ID NO: 5) residues tagged at the C-terminus was developed which resulted in 3-4 fold yield compared to that of the original protein after purification. The resultant recombinant protein (TAT-HOXB4H) contains 6 histidine (SEQ ID NO: 5) residues at the C-terminus. This method was described in detail in the PCT application PCT/CN2006/000646.
It was shown that the recombinant TAT-HOXB4H protein can be used to expand human peripheral blood or UCB stem cells and the expanded stem cells still possess their pluripotency. Furthermore, the stem cells treated with the recombinant TAT-HOXB4H protein incorporated into the bone marrow of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice and human leukocytes were detected in peripheral white blood cells, indicating immune and hematopoiesis reconstitution in the mice.
However, recombinant TAT-HOXB4H proteins have never been used before as a stimulator of hematopoiesis in vivo, specifically, to enhance hematopoietic reconstitution, expansion, bone marrow re-population and to increase the number of peripheral circulating stem cells, particularly after chemotherapy or irradiation. Krosl et al. (2003) and Amsellem et al. (2003) were not able to obtain large amounts of highly stable HOXB4 protein to be used in clinical studies to expand HSCs. In the present invention, the total amount of TAT-HOXB4H protein obtained after purification generally ranges from 6-10 mg from a 1 liter culture, while the total amount of TAT-HOXB4 protein obtained after purification from a 1 liter culture using prior art methods generally ranges from 1-2 mg. The pTAT-HA-HOXB4 plasmid used to express the TAT-HOXB4 protein using prior art methods was a gift from Dr. Guy Sauvageau, University of Montreal, Canada. The method of purifying the TAT-HOXB4H protein using the present invention clearly indicates the increased yield of protein necessary for the in vivo administration. Krosl et al. (2003) also reported that most of their TAT-HOXB4 protein was lost after 4 h of incubation in medium with serum. The present invention shows a significantly high stability of TAT-HOXB4H protein even after 4 weeks, which is a key factor for the use of TAT-HOXB4 protein in clinical studies.